Xylanase is an enzyme which degrades xylan. It is a useful enzyme employed in a pretreatment process for the bleaching of pulp in paper manufacturing or in the manufacturing of functional xyloligosaccharides.
Since Viikari et al. reported that the bleaching effect on kraft pulp is improved with xylanase Abstract of the Presentations for the 3rd International Conference on Biotechnology in the Pulp and Paper Industry (1986), p. 67!, xylanase has begun to attract attention in the paper pulp industry. In the process of pulp manufacturing, there are many steps, such as kraft digesting and bleaching, which carried out under high temperature conditions. Accordingly, in order to efficiently use xylanase in these steps, a highly thermostable xylanase is desired. Since the use of a thermostable xylanase enables an enzyme reaction at a high temperature, facilities and energy required for cooling can be reduced. Furthermore, the contamination during the enzymatic treatment can also be prevented.
On the other hand, it is obvious that a microorganism which produces a high amount of an enzyme is advantageous from the view point of cost reduction in enzyme production.
Therefore, a highly thermostable xylanase and a microorganism which produces a high amount of this enzyme are desired.
With respect to microorganisms producing a xylanase, a number of microorganisms such as filamentous fungi belonging to the genera Aspergillus, Trichoderma, Aureobasidium and Schizophyllum commune and bacteria belonging to the genera Bacillus, Clostridium and Streptomyces according to a review of xylanase (Wong et al., Microbiological Reviews, September, 305, 1988) and the like are known. The reaction pH of the xylanases from these microorganisms is acidic to neutral and their reaction temperature ranges from 40 to 80.degree. C. There are also known microorganisms producing an alkali xylanase which has an activity in the alkaline side. For example, microorganisms belonging to the genus Bacillus (Honda et al., System. Appl. Microbiol., 8, 152, 1986; Okazaki et al., Appl. Microbiol. and Biotechnol., 19, 335, 1984), the genus Aeromonas (Ohkoshi et al., Agric. Biol. Chem., 49, 3037, 1985) and the genus Streptomyces (Vyas et al., Biotechnol. Let., 12, 225, 1990) are known.
Among these xylanase-producing microorganisms, a number of filamentous fungi also produce cellulase as well as xylanase. Therefore, if used in pulp and paper manufacturing processes, such cellulase would cause various problems such as decrease in pulp yield or paper strength. Furthermore, filamentous fungi require a longer cultivation period than bacteria. On the other hand, the xylanase productivity of those bacteria is rather low.
Recently, Viikari et al. reported that, using alkalitolerant Bacillus circulans VTT-E-87305 strain, they obtained the highest xylanase activity (400 U/ml) within two days at pH 8-8.5 at 30.degree. C. (Appl. Microbiol. Biotechnol., 37, 470, 1992). However, the thermostability of this xylanase is not clear. In addition, since the cultivation temperature is as low as 30.degree. C., it is difficult to use this enzyme for an enzyme reaction at a high temperature.
On the other hand, with respect to the purification of a thermostable xylanase produced by a microorganism belonging to the genus Bacillus, there have been reported W1-I thermostable xylanase having a molecular weight of 21,500, an isoelectric point at 8.5, an optimum pH for reaction of 6.0 and an optimum temperature for reaction of 65.degree. C. and W2-I thermostable xylanase having a molecular weight of 22,500, an isoelectric point at 8.3, an optimum pH for reaction of 6.0 and an optimum temperature for reaction of 65.degree. C. derived from basophilic, thermophilic Bacillus W-1 and W-2, respectively (Okazaki et al., Agric. Biol. Chem., 49, 2033, 1985).
With respect to Bacillus stearothermophilus which is known as a thermophilic Bacillus, T. Nanmori et al. reported the purification and production of a thermostable xylanase having a molecular weight of 39,500, an isoelectric point at 5.1, an optimum pH for reaction of 7.0 and an optimum temperature for reaction of 60.degree. C. from a culture filtrate of Strain 21 (J. Bacteriol., 172, 6669, 1990). However, the amount of activity for two days at 55.degree. C. is only 1.96 U/ml.
In addition, with respect to applications of xylanase, there have been reported attempts to reduce bleaching chemicals and AOX (adsorbable organic halogen compounds, especially organic chlorides) by treating pulp with xylanase (e.g., Japanese Unexamined Patent Publications No. 2-210085, No. 2-210086, No. 2-221482, No. 2-264087, No. 2-293486, No. 3-40887 and No. 3-505785; L. S. Pederson et al., Production of Bleached Chemical Pulp in the Future International Pulp Bleaching Conference, Vol. 2, 107, 1991; KAMI PARUPU GIJUTU TAIMUZU (Paper Pulp Technology Times) issued on May 20, 1992; S. Hogman et al., Biotechnology in Pulp and Paper Industry, Uni Publishers Co., Ltd., p. 107, 1992; and Viikari et al., Biotechnology in Pulp and Paper Industry, Uni Publishers Co., Ltd., p. 101, 1992).
In these attempts, however, while a high temperature treatment at 40-100.degree. C. is necessary for the bleaching step in the pulp and paper manufacturing processes, a non-thermostable enzyme is often used in the bleaching process. For this enzymatic treatment, pulp must be cooled to the optimum temperature for the enzyme reaction and then heated for the subsequent step, which requires enormous energy.
Because of high temperature cultivation, this enzyme can be produced without cooling facilities or with saving of cooling water, and yet with reducing the possibility of pulp contamination with various microorganisms. It is possible to manufacture the enzyme at a low cost. Accordingly, a thermostable xylanase is desired.
In addition, it is also desired to obtain a thermostable xylanase in large quantity by expressing the gene by recombinant DNA techniques and a gene coding for the xylanase.
A number of papers have been reported on xylanase genes. For example, there are genes derived from bacteria, such as Bacillus circulars Yang R. C. A. et al., Nucleic Acids Res. 16:7187-7187 (1988)!, Bacillus subtilis Paice M. G. et al., Arch. Microbiol. 144:201-206 (1986)!, Pseudomonas fluorescens Kellett L. E. et al., Biochem. J. 272:369-376 (1990)! and Ruminococcus flavefaciens Zhang J. X. et al., Mol. Microbiol. 6:1013-1023 (1992)!, and genes derived from fungi, such as Clostridium acetobutylicum Zappe et al., Nucleic Acids Res. 18:2179-2179 (1990!, Aspergillus awamori Ito K. et al., Biosci. Biotechnol. Biochem. 56:1338-1340 (1992)! and Streptomyces lividans Shareck F. et al., Gene 107:75-82 (1991)!. However, it is not clear whether those enzymes produced by the transformants obtained by using these genes are suitable for bleaching or not.